tolerance tests. To test enteral effect of uridine,
uridine was dissolved together with glucose in
H2O and the glucose-uridine solution (0.25 g/ml
glucose and 0.1 g/ml uridine in H2O) was used
for oral gavage at the same dose as glucose tolerance test.

PALA (100 mg/ml in 0.9% saline) was administrated via intraperitoneal injection at 62.5 mg/kg
body weight to ob/ob (10 to 35 weeks old) or
male WT mice (16 to 20 weeks old on HFD for
30 days). Food was removed after PALA injection up to 24 hours to monitor response in body
temperature and plasma glucose. PALA was synthesized at UT Southwestern Medical Center as
described (45) with purity greater than 95% as
assessed by nuclear magnetic resonance. STZ
(Sigma) was dissolved freshly in ice-cold sodium
citrate buffer (0.1 M, pH 4.5) and administrated
via intraperitoneal injection to male C57BL/6
mice (10 to 15 weeks old) at 120 mg/g body weight
as described previously (46). Body weight and
plasma glucose levels were monitored up to
7 weeks post STZ treatment, and the mice were
euthanized for plasma, bile, and tissue collection
after 24 hours of fasting.

RNA isolation and qPCR

Male C57BL/6 mice (15 to 25 weeks old) fed or
fasted of 24 hours were euthanized and tissues
were harvested and immediately frozen in liquid
nitrogen. Total RNA was isolated using NucleoSpin
RNA II mini columns (Macherey-Nagel) from 50
to 100 mg tissue and total RNA (100 ng to 1 mg)
was used for reverse transcription with iScript
kit (Bio-Rad). cDNA samples were then diluted
tenfold with ddH2O and stored at –20°C for qPCR
(Roche). Primers are shown in table S2. 18S RNA
served as an internal control.

Statistical analysis

Results are reported as mean ± SEM. Statistical analysis of the data was performed with
two-tailed Student t test or two-way ANOVA,
as specified. Paired t test was performed for
samples collected from the same mouse during
a time course study. A P value < 0.05 was considered as significant. Statistical software consisted
of Microsoft Excel and GraphPad Prism 6.04.